ELISA using an antibody to a specific protein is known as a method of quantifying a specific protein contained in a trace amount in a measurement sample. However, in order to prepare the antibody, it is necessary to prepare a highly pure peptide or protein serving as an antigen, and to prepare an antiserum by administering the peptide or protein to an animal, and the procedure is thus very troublesome. In addition, an antibody may react with a highly homologous protein, and hence it is necessary to examine whether only a specific protein is measured.
In addition, reactivity of a protein with an antibody varies due to thermal denaturation and the like. Therefore, in a sterilized product, such as food, it is impossible to determine the protein content with high accuracy even if a specific protein is measured by ELISA described above.
Quantitative analysis methods for proteins, which have developed rapidly in recent years, include a method using a high performance liquid chromatogram tandem mass spectrometer (hereinafter, referred to as LC/MS/MS). A method has been developed for identifying a protein, which involves performing separation of a sample containing many kinds of proteins by two-dimensional electrophoresis and measuring peptides by LC/MS/MS obtained by an enzymatic treatment of the resultant spots. If measurement is performed by LC/MS/MS, There are the following advantages: it is possible to reduce the steps of pretreatment because derivatization is unnecessary, which is required in conventional GC/MS; and it is possible to measure a polymer compound such as a protein or peptide. In a method of identifying a protein by LC/MS/MS, the protein becomes able to be identified by: determining the mass of a peptide fragment produced from a sample protein using a specific protease by the first MS; fragmenting the peptide; performing the second MS to anticipate the amino acid sequence of the peptide; and comparing all the anticipated peptide sequences with a database.
As the method of quantifying a protein by LC/MS/MS, a method which involves labeling an amino acid in a target peptide with deuterium and measuring the amino acid has been reported (see Non-Patent Document 1, for example). However, in this method, the deuterium-labeled amino acid is used as internal standard substances, and because the deuterium-labeled amino acid is very expensive and rare, the peptide synthesis using the method may limit the application thereof.
In addition, a method of detecting an animal-derived protein in a complex mixture by LC/MS/MS is disclosed (see Patent Document 1, for example). However, this method has a difficulty in removing interfering substances and maintaining the level of contaminants at a low level and also requires cumbersome procedures.
There is required a measurement method which can quantify a specific protein or peptide contained in a trace amount with high accuracy and ease, and even without using any expensive reagent.    [Patent Document 1] JP 2005-513481 B    [Non-Patent Document 1] David R. Barnidge et al., Analytical Chemistry, Vol. 75, No. 3, 2003